A very important part of our laboratory effort is to identify unique cell surface markers of KSC in order to distinguish these stem cells from other keratinocytes. Currently, unique cell surface markers have not been identified for either keratinocyte stem cells or other epithelial tissues. The ability to identify and manipulate keratinocyte stem cells is important for two reasons. First, it will allow us to gene target these cells, which is important for long-term gene expression in a renewing tissue. Second, the epidermis and other epithelial are sources for a majority of human cancer and the ability to selectively target therapies to "cancer stem cells" of these tissues may be critical to effectively treat epithelial-derived cancer. Keratinocyte stem cells are best identified by their ability to retain a BrdU label, and we have successfully FACS sorted pure populations of these "label retaining" cells or LRC. (Unfortunately, the detection of BrdU requires that these cells be fixed and permeablized, making them unsuitable for biological studies requiring living cells.) Good progress has been made in identifying membrane proteins in our ongoing collaboration with the Biomedical Proteomics Program at Frederic and we are now working to quantitatively assess and compare the plasma membrane proteins prepared from LRC keratinocytes to control keratinocyte populations with high throughput mass spectrometry (MS) analysis in order to identify unique cell surface markers. Recently, a population of very primitive hematopoietic stem cells (side population or SP cells) have been identified because of unique fluorescent emission characteristics due to their ability to exclude HO33342 nuclear dye. We have recently described a SP population of keratinocytes and are currently trying to determine if these SP keratinocytes function as stem cells in vivo and possess long-term repopulating ability, using our in vivo stem cell assay. We have been using microarray analysis of gene expression in these and control keratinocytes in order to try to understand their function in the skin. Patterns of gene expression in SP keratinocytes can be also be assessed on a protein level, and these SP cells can also be analyzed by high-throughput mass spectrometry for the presence of unique cell surface markers. The experimental approaches we have developed to identify cell surface markers of keratinocyte stem cells can also be applied to cancer stem cells, and the in vivo models we have developed to assess keratinocyte stem cell behavior will be critical in determining if cancer stem cells are present in skin cancers (squamous cell carcinoma and basal cell carcinoma) and in other epithelial cancers. The identity and characterization of cancer stem cells may be required before effective therapies can be developed to treat epithelial cancer.